1. Field of the Invention
The present invention relates to a method for producing a DNA chip (DNA microarray) in which several thousands to not less than ten thousand kinds of different types of DNA fragments are aligned and fixed as spots at a high density on a base plate such as a glass microscope slide.
2. Description of the Related Art
Methods for analyzing genetic structure have been remarkably progressed in recent years. A large number of genetic structures represented by those of human genes have been clarified. The analysis of the genetic structure as described above uses a DNA chip (DNA microarray) in which several thousands to not less than ten thousand kinds of different types of DNA fragments are aligned and fixed as spots on a base plate such as a glass microscope slide.
Those widely used as a method for forming the spots during the production of the DNA chip are based on a system such as the QUILL system, the pin and ring system, and the spring pin system in which a sample solution containing DNA fragments is supplied (stamped) onto the base plate by using a so-called pin. Even when any one of the foregoing methods is adopted, it is necessary to suppress the dispersion of the volume and the shape of each of the spots to be low so that the distance between the respective spots is maintained to be constant.
On the other hand, in order to realize a higher density, it is also greatly expected to develop a new method in which the shape control performance is satisfactory for the spot, and the productivity is excellent.
As shown in FIG. 16, when a spot is formed by dripping a sample solution onto a base plate 200, the spot is hemispherical in accordance with the surface tension. In this procedure, a substantial amount of the sample immobilized on the base plate 200 resides in a slight portion 204 contacting with the base plate 200. The amount is merely a part of the whole (spherical matter). The remaining portion 206 is not immobilized, and hence it is washed away during the washing step performed thereafter. As a result, a problem arises that a large amount of the sample solution is lost, and the efficiency of the use of the sample solution is low.
The cost for the production of the DNA chip is substantially determined by the amount of the sample solution. In the case of the procedure described above, almost all of the sample solution is washed away, and the procedure is disadvantageous in view of the production efficiency.
Several thousands to not less than ten thousand kinds of different types of sample solutions are dripped onto one base plate. However, the viscosity and the surface tension differ for each of the different types of the sample solutions. Therefore, in order to obtain an identical spot diameter, it is necessary to change the dripping amount of the sample solution depending on, for example, the viscosity and the surface tension.
However, in the case of the conventional technique, the sample solution adhered to a pin is allowed to physically make contact with the base plate together with the pin so that the sample solution is dripped. Therefore, the spot is formed on the base plate by means of one time of dripping. As a result, the following problem arises. That is, it is impossible to perform any delicate control of the dripping (control of the dripping amount and the dripping position), and dispersion occurs in the spot diameter formed on the base plate.
In order to more reliably immobilize the DNA fragment in the sample solution onto the base plate, a method has been also developed, in which an organic or inorganic polymer is mixed in the sample solution to physically hold the DNA fragment in the polymer cross-link. However, in the case of this procedure, the following problem arises. That is, the viscosity of the sample solution is increased, and the sample solution tends to be dried, thickened, and solidified. The pot life of the sample upon the formation of the spot is shortened, and the amount of one time of dripping is increased.
The present invention has been made taking the foregoing problems into consideration, an object of which is to provide a method for producing a DNA chip, which makes it possible to improve the efficiency of the use of an expensive sample solution, improve the productivity of the DNA chip, and improve the yield.
Another object of the present invention is to provide a method for producing a DNA chip, which makes it possible to control the supply depending on the type of a sample solution supplied onto a base plate, realize a uniform spot diameter formed on the base plate, and improve the reliability and the quality of the DNA chip.
According to the present invention, there is provided a method for producing DNA chip including a large number of spots of sample solutions arranged on a base plate, the method comprising the step of supplying the sample solutions onto the base plate; wherein the sample solution is supplied a plurality of times to form one of the spots.
Accordingly, it is possible to improve the yield of the DNA chip. In this process, it is preferable that the sample solution is supplied by an ink-jet system.
When the ink-jet system is used, a large number of liquid droplets in a required amount can be supplied onto the base plate at a high speed (to 100 kHz) in a manner to make no contact with the base plate. The supply source is continuously supplied via a pouring port and a cavity connected to a discharge port for discharging the liquid droplets. Therefore, unlike the conventional pin system, it is unnecessary to move the pin to a supply source (sample well) for the sample solution to immerse the pin tip in the sample solution every time the spot is formed. Thus, it is possible to form the spots on a large number of base plates in a short period of time.
It is preferable that the sample solution is obtained by diluting a sample containing a DNA fragment to give a predetermined concentration. In this process, it is preferable that the sample solution is obtained by diluting the sample containing the DNA fragment with water or an aqueous solution containing sodium chloride or an aqueous solution containing a polymer. It is preferable that the sample solution is diluted to give a concentration of such a degree that final desired base pairs per one spot are satisfied, by performing the supply a plurality of times to form one of the spots.
The following advantage is obtained by diluting the concentration of the sample. That is, it is possible to relatively decrease the amount of the expensive DNA fragment in the sample solution adhered or remained in the supply flow passage path at the stage at which the supply of the sample solution onto the base plate comes to an end. The following effect is also obtained. That is, it is possible to avoid the occurrence of any defect which would be otherwise caused such that the solution is dried, thickened, and solidified due to the concentrated sample solution, and the discharge port is clogged to cause defective discharge. A greater advantage is obtained that when the sample solution is supplied onto the base plate, then the sample solution becomes not hemispherical but flat. In this case, almost all of the sample solution supplied to the base plate is immobilized on the base plate. Therefore, most of the sample solution is not washed away during the washing step to be performed thereafter. Thus, it is possible to improve the efficiency of the use of the sample solution.
Further, it is possible to realize a uniform spot diameter of the sample solution formed on the base plate, by changing the degree of the dilution depending on the type of the DNA fragment contained in the sample solution so that the viscosity and the surface tension of the sample solution are varied.
Further, it is preferable that the sample solution is diluted with the aqueous solution containing the polymer. Accordingly, the shape-retaining performance is increased for the spot shape after being supplied onto the base plate. The shape is stabilized, and it is possible to avoid any change of the shape which would be otherwise caused by drying and contraction of the spot.
As described above, according to the present invention, it is possible to improve the efficiency of the use of the expensive sample solution, and it is possible to improve the productivity of the DNA chip and improve the yield. The dripping control can be performed depending on the type of the sample solution to be dripped. It is possible to realize the uniform spot diameter formed on the base plate. It is possible to improve the reliability and the quality of the DNA chip.
In the production method described above, the sample is prepared by carrying out the steps of PCR-amplifying the DNA fragment to prepare a PCR product; drying the PCR product to obtain DNA powder; and dissolving the DNA powder in a buffer solution. The sample as described above undergoes no change in quality, and it is well dispersed in the aqueous solution, when the sample is diluted. The sample is suitable for the dilution, and the concentration can be correctly managed upon the dilution.
In the production method described above, it is also preferable that a dispenser is used when the sample solution is supplied onto the base plate, the dispenser comprising a plurality of arranged micropipettes each including a pouring port for pouring the sample solution from the outside, a cavity for pouring and charging the sample solution thereinto, and a discharge port for discharging the sample solution, formed on at least one or more substrates, the micropipette further including a piezoelectric/electrostrictive element disposed on at least one wall surface of the substrate which forms the cavity so that the sample solution is movable in the cavity, and mutually different types of the sample solutions being discharged from the discharge ports of the respective micropipettes.
Every time the piezoelectric/electrostrictive element is driven, a minute amount of the liquid is discharged from the discharge port, and the volume thereof is minute and constant without dispersion. The driving cycle can respond to the high frequency wave by using the piezoelectric/electrostrictive element. The time required for the discharge is shortened as well. The sample solution is moved in the closed space from the pouring of the sample solution to the discharge. Therefore, the sample solution is not dried during any intermediate process. Further, the entire substrate can be formed to be small and compact. Therefore, it is possible to shorten the flow passage through which the sample solution is moved. Accordingly, it is possible to suppress the problem of adhesion of the sample solution to the flow passage wall to be minimum, and it is possible to avoid the deterioration of the efficiency of the use of the sample solution.
It is also preferable that a dispenser is used when the sample solution is supplied onto the base plate, the dispenser comprising a plurality of arranged micropipettes each including a pouring port for pouring the sample solution from the outside, a cavity for pouring and charging the sample solution thereinto, and a discharge port for discharging the sample solution, formed on at least one or more substrates, so that the sample solution is movable in the cavity, and the sample solution of an identical type is discharged from at least two or more of the discharge ports to form one spot.
When the sample solution of the identical type is discharged from two or more of the discharge ports to form one spot, it is possible to increase the speed for forming the spot, and it is possible to improve the throughput.
In general, when the sample solution is supplied a plurality of times, the spot diameter is increased every time the supply is performed. However, the number of times of supply can be increased without increasing the spot diameter by delaying (increasing) the supply interval, or applying a treatment so that the sample solution supplied onto the base plate is quickly dried, thickened, and solidified as described later on.
When the supply interval is increased, the sample solution, which is disposed at the portion of the discharge port opened toward the discharge side, is dried to some extent before the discharge, and the sample solution is discharged in a state in which the viscosity is increased, i.e., in a so-called semidried state. Therefore, the spot diameter is not increased even when the supply is repeated. However, in this method, the time required to form the spot is consequently increased, which is not preferred. In such a case, there are a lot of limitations to periodically manage the so-called semidried state, and the nozzle of defective discharge is apt to appear.
Accordingly, when the sample solution of the identical type is discharged from the two or more discharge ports to form one spot, two or more discharge ports, at which the sample solution is progressively dried at the portion of the discharge port opened toward the discharge side, exist during the period in which the discharge port is moved to the discharge position, i.e., during the waiting period until the discharge is started. The identical sample solution can be supplied by using the discharge ports as described above. As a result, it is possible to shorten the time required to form the spot.
If the nozzle of defective discharge appears by any chance, it is possible to prevent the loss of the expensive sample solution beforehand, by discharging with a functioning nozzle. Further, it is preferable to use a structure in which one pouring port communicates with the cavity connected with at least two or more of the discharge ports for discharging the sample solution of the identical type, because it is possible to reduce the number of pouring operations for the sample solution.
It is also preferable that the sample solution of the identical type is discharged at substantially the same time from at least two or more of the discharge ports, in the case of using the non-contact ink-jet system. In this case, it is also preferable that the falling points are allowed to coincide with each other for the sample solution to be discharged. By doing so, it is possible to improve the speed for forming the spot.
However, in order to obtain a higher accuracy for the position accuracy of the sample solution to be supplied onto the base plate, it is preferable to use a method in which the sample is supplied at deviated discharge timings when each of the discharge ports is located just over the spot formation position.
It is preferable that the plurality of micropipettes, each of which is constructed such that the sample solution is moved in the cavity in a laminar flow, are arranged. When the sample solution is moved in the form of laminar flow, it is possible to avoid the occurrence of bubbles or the like, and it is possible to avoid defective discharge. Thus, the durability of the micropipette is increased.
It is preferable that, when the dispenser is used, mutually different types of the sample solutions are poured into the plurality of cavities from the pouring ports corresponding to the discharge ports for discharging the mutually different types of the sample solutions, and then the different types of the sample solutions in the plurality of cavities are discharged from the discharge ports by driving the piezoelectric/electrostrictive elements. According to the arrangement as described above, the plurality of different types of the sample solutions can be supplied onto the base plate at the same timing without causing any cross-contamination.
It is also preferable that, when the dispenser is used, a substitution solution is previously charged into the plurality of cavities, different types of the sample solutions are subsequently poured from the pouring ports while effecting substitution in the plurality of cavities, and then the piezoelectric/electrostrictive elements are driven so that the different types of the sample solutions in the plurality of cavities are discharged from the discharge ports. The occurrence of the defective discharge can be completely avoided, and the expensive sample can be efficiently discharged by previously substituting the interior of the cavity with the inexpensive substitution solution, and then effecting the substitution with the expensive sample.
The substitution from the substitution solution to the sample solution in the cavity may be performed by aspirating and discharging the substitution solution from the discharge port, for example, by means of vacuum suction. However, it is preferable that the different types of the sample solutions are poured from the pouring ports while effecting the substitution in the plurality of cavities, while driving the piezoelectric/electrostrictive elements. By doing so, the amount of the substitution solution to be discharged can be accurately controlled, without involving any loss of the discharge of the expensive sample solution.
The end point of the completion of the substitution may be controlled, for example, by the substitution time and the discharge amount by previously determining the volume and the movement speed of the sample. However, it is more preferable that the end point of the completion of the substitution is recognized by sensing the change of the fluid characteristic in the concerning cavity, because the end point can be detected more accurately.
In the present invention, the completion of the substitution is recognized by sensing the change of the fluid characteristic in the cavity. Therefore, even when the sample solution and the substitution solution are mixed with each other to some extent in the flow passage, the mixed portion can be easily distinguished from the unmixed portion to make accurate judgement. As a result, it is possible to decrease the amount of the sample solution which should be purged by being mixed with the substitution solution. Thus, it is possible to increase the efficiency of the use of the sample solution.
The change of the fluid characteristic in the cavity may be recognized by applying a voltage to the piezoelectric/electrostrictive element to excite vibration, and detecting the change of the electric constant caused by the vibration. Accordingly, it is unnecessary to install, for example, any special detecting element. The detection can be performed inexpensively and accurately.
It is preferable that the substitution solution is previously subjected to a degassing treatment. By doing so, the substitution solution can be smoothly charged into the cavity without generating any bubbles or the like and without causing any clogging during the process. Accordingly, the substitution into the sample solution is reliably performed, and the discharge is stabilized. Further, it is also preferable that the substitution solution is poured and charged into the cavities, an intermediate solution containing no DNA fragment, which has approximately the same specific gravity as that of the sample solution, is subsequently poured from the pouring ports to effect substitution in the cavities, the different types of the sample solutions are subsequently poured from the pouring ports into the cavities, and thus the sample solution are charged. The inexpensive intermediate solution containing no DNA fragment, which has approximately the same specific gravity as that of the sample solution, is allowed to intervene between the substitution solution and the sample solution. Accordingly, it is possible to avoid an inconvenience which would be otherwise caused such that the expensive sample solution is mixed with the substitution solution having the different specific gravity, and consequently the purge amount is inevitably increased.
In the present invention, a plurality of micropipettes are used. Therefore, many kinds of samples can be simultaneously supplied at once. Further, any pipette, in which any partial defect occurs, can be exchanged with ease. Therefore, it is easy to perform the maintenance. Further, the discharge ports are aligned and arranged two-dimensionally. Therefore, for example, this arrangement is optimum when the spots are aligned and fixed two-dimensionally on the base plate.
In the present invention, it is preferable that when the sample solution is supplied onto the base plate, the sample solution is supplied while drying, thickening, or solidifying at least the sample solution. Accordingly, the immobilization of the sample solution supplied onto the base plate is quickened. It is possible to effectively avoid any weakening (phenomenon in which the spot diameter is expanded) associated with the dilution of the sample solution.
It is exemplified that the treatment of drying, thickening, or solidifying the sample solution is, for example, to heat the base plate, and to heat the discharged or supplied sample solution. It is preferable to use, for example, a laser beam, an infrared ray, and an electromagnetic wave, as a heating method.
The methods as described above especially make it possible to selectively heat a minute region. As in the present invention, it is necessary that the spot of the discharged sample solution is quickly heated, while it is necessary to avoid the occurrence of the defective discharge due to the drying or the like caused by the heating at the discharge port disposed just closely to the spot. In such a situation, it is preferable to use the treatment of drying, thickening, or solidifying the sample solution as described above. Especially, the electromagnetic wave can be reliably cut off by means of a metal shield. Therefore, the electromagnetic wave is preferred in order to avoid any unnecessary heating of the discharge port. When the laser beam or the infrared ray is used, the laser beam or the infrared ray is radiated onto the base plate to indirectly heat the sample solution.
In the present invention, it is also preferable that a treatment of drying, thickening, or solidifying the dripping sample solution is to cool the base plate or the discharged or supplied sample solution. The cooling procedure is preferably adopted when DNA in the sample solution is damaged by the heating or when any component in the sample solution is softened by the heating.
In the present invention, it is also preferable that when the sample solution is supplied onto the base plate, the sample solution is supplied while deviating a supply position, or the sample solution is supplied while changing a supply amount. That is, for example, the sample solution is supplied to mutually different positions two times to one hundred times to form one spot diameter depending on the type of the sample solution. In this procedure, the number of supply operations can be changed depending on the type of the sample solution, and the supply position can be determined. Therefore, all of the spot diameters can be formed to be uniform, regardless of the type of the sample solution. Thus, it is possible to improve the DNA chip and improve the reliability.
The formation of one spot while deviating the supply position cannot be performed by using the conventional pin system spotting. The technique as described above can be firstly realized in accordance with the ink-jet system in which the supply amount per one droplet is about {fraction (1/100)} to {fraction (1/10)} as compared with the pin system. When the technique is combined with the treatment of drying, thickening, or solidifying the sample solution, it is possible to realize a spot shape other than the conventional circular spot shape. An advantage is obtained such that it is possible to widen the range capable of effecting the matching with a DNA chip reader (for example, a CCD image pickup device).
Further, the formation of one spot while deviating the supply position with minute droplets also makes it possible to control the shape of the spot in the height direction by adjusting the stacking position thereof. An advantage is obtained such that the fluorescence intensity pattern emitted from the spot can be freely designed in the spot.
The supply amount can be changed by changing the number of supply operations, as well as by changing the discharge condition, i.e., the voltage pattern applied to the piezoelectric/electrostrictive element in the case of the ink-jet system.
In the present invention, it is preferable that vibration is applied to the sample solution during the supply or prior to the supply of the sample solution onto the base plate.
In this procedure, it is possible to avoid any precipitation of the DNA fragment contained in the sample solution. It is possible to uniformly disperse the DNA fragment in the sample solution. Accordingly, it is possible to almost exclude the dispersion of the content of the DNA fragment for the same types of the sample solutions to be formed on the respective base plates. It is possible to exclude the dispersion in the genetic analysis for every base plate.
According to another aspect of the present invention, there is provided a method for producing a DNA chip including a large number of spots of sample solutions arranged on a base plate, the method comprising the step of supplying the sample solutions onto the base plate in accordance with an ink-jet system, wherein when the sample solution is supplied onto the base plate, a humidity around a portion to which the sample solution is discharged and supplied is selectively increased as compared with that around the other portions so that the sample solution is not dried, thickened, or solidified. Accordingly, especially when the sample solution, which tends to be dried, thickened, or solidified, is used, it is possible to avoid any defective discharge.
In the present invention, it is also preferable to adopt the steps of cooling the base plate to be not more than 0xc2x0 C. after preparing the base plate on which the large number of spots are arranged by supplying the sample solutions onto the base plate, and then returning the base plate in an atmosphere at room temperature in which a sufficient volume of gas exists at a humidity of not less than 30%.
It is also preferable that the base plate is exposed in an atmosphere in which a sufficient volume of gas exists at a humidity of not less than 80%, or to water vapor containing mist, after preparing the base plate on which the large number of spots are arranged by supplying the sample solutions onto the base plate.
The inventions described above are preferably adopted when the sample solution, in which a polymer or the like is mixed to increase the viscosity, is used, the dripping method for the sample solution is adjusted, and the shape of the spot on the base plate exhibits, for example, a so-called doughnut-shaped configuration in which the circumferential edge portion is bulged, and the central portion is recessed.
In the case of the doughnut-shaped configuration as described above, the boundary between the base plate and the circumferential edge portion of the spot is conspicuous, and it is easily observed. When the spot of the colorless and transparent liquid such as the sample solution containing the DNA fragment is formed, for example, on the colorless and transparent glass base plate, the following advantage is obtained. That is, it is easy to observe the shape of the spot, and it is easy to inspect whether the shape of the spot is satisfactory or defective.
However, in the case of the spot having the doughnut-shaped configuration as described above, the substantial immobilized sample is plentiful (thick) at the circumferential edge portion, even when most of the bulged portion at the circumferential edge is washed away in the washing step during the immobilization to be performed thereafter. Therefore, in the case of the use as the DNA chip, the distribution of fluorescence emission amount emitted from the spot exhibits a doughnut-shaped configuration in the spot, consequently causing a factor to bring about the dispersion and the deterioration of the sensitivity.
Therefore, in order to realize easy inspection (doughnut-shaped configuration) and good spot shape (non-doughnut-shaped configuration), the following method is appropriate. That is, when the sample solution is supplied onto the base plate, the discharge is performed in accordance with the ink-jet system or the like to drip the sample solution onto the base plate so that the sample solution is concentrated at the circumferential edge portion of the spot by controlling the kinetic energy and the hydrophobic property with respect to the base plate to form the doughnut-shaped configuration. After that, the viscosity of the sample solution is previously increased to such an extent that the spot is not spherical against the surface tension of the liquid, while the fluidity of the sample solution to form the spot is increased after completion of the inspection so that the doughnut-shaped configuration is changed to the non-doughnut-shaped configuration by the aid of the surface tension. The present invention is preferred in order to realize the method described above.
That is, the base plate, on which the spot having the doughnut-shaped configuration is formed, is cooled to be not more than 0xc2x0 C., and then the base plate is returned in the atmosphere at room temperature in which the sufficient volume of gas exists at the humidity of not less than 30%. Alternatively, the base plate is exposed in the atmosphere in which the sufficient volume of gas exists at the humidity of not less than 80%, or to the water vapor containing the mist. By doing so, the water is incorporated into the sample solution from the surrounding gas, or the mist makes contact to increase the fluidity of the sample solution. Accordingly, the shape of the spot is changed to a hemispherical configuration which is the non-doughnut-shaped configuration. Thus, it is possible to improve the sensitivity of the DNA chip and reduce the dispersion of the sensitivity. Of course, after the shape of the spot is hemispherical, the base plate may be immediately introduced into the drying step to fix the shape. It is a matter of course that when the base plate is exposed to the water vapor, it is necessary that the temperature of the water vapor is not more than a temperature of such an extent that the DNA fragment is not denatured.
The above and other objects, features, and advantages of the present invention will become more apparent from the following description when taken in conjunction with the accompanying drawings in which a preferred embodiment of the present invention is shown by way of illustrative example.